Fas-based d1OS-mediated cytotoxicity requires macromolecular synthesis for effector cell activation but not for target cell death

1994 ◽  
Vol 345 (1313) ◽  
pp. 303-309 ◽  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Surface ◽  
Target Cell ◽  
Cell Activation ◽  
Effector Cell ◽  
Apoptotic Cell ◽  
Target Cells ◽  
Macromolecular Synthesis ◽  
Effector Cells

Two main mechanisms seem at play in T cell-mediated cytotoxicity, a process in which target cell death often follows an apoptotic cell death pattern. One of these involves Fas at the target cell surface and a Fas ligand at the effector cell surface. This allowed us to reinvestigate the long-standing question of macromolecular synthesis requirement in T cell-mediated cytotoxicity, using the dlOS model cell line which is cytotoxic apparently only via the Fas molecularly defined mechanism. We showed, first, that induction of cytotoxic activity of effector cells, obtained by preincubating these effector cells with a phorbol ester and a calcium ionophore, could be inhibited by macromolecular synthesis inhibitors (cycloheximide, actinomycin D, DRB). We then investigated whether macromolecular synthesis was required, when effector and target cells were mixed, to obtain target cell death. Preincubating already activated effector cells for 30 min with macromolecular synthesis inhibitors, then adding target cells and performing the 51 Cr release cytotoxicity test in the presence of these inhibitors, did not significantly decrease subsequent target cell death, indicating that already activated effector cells could kill without further requirement for macromolecular synthesis. In addition, target cell preincubation for up to 3 h in the presence of one of these inhibitors did not decrease cell death. The high sensitivity of mouse thymocytes to this type of cytotoxicity enabled us to devise the following experiment. As previously shown by others, thymocyte death induced by dexamethasone (DEX) could be blocked by coincubation with cycloheximide (CHX). Such DEX-treated CHX-rescued thymocytes, the survival of which was an internal control of efficiency of protein synthesis inhibition, were then subjected to effector cells in the presence of CHX, and were shown to die. Thus, there is no requirement for macromolecular synthesis at the target cell level in this variety of apoptotic cell death. Altogether, in this defined mechanism of T cell-mediated cytotoxicity, macromolecular synthesis is required for dlOS effector cell activation, but not for lysis by already activated effector cells nor for target cell death.

scholarly journals In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2763-2763
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Michael Weichel ◽  
Kristina Ellwanger ◽  
...  
Keyword(s):  
T Cell ◽  
Target Cell ◽  
Binding Sites ◽  
Effector Cell ◽  
Nk Cell ◽  
Target Cells ◽  
Tumor Target ◽  
Effector Cells

Abstract Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12. Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed. Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described. Conclusions: Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

1979 ◽  
Vol 150 (6) ◽  
pp. 1310-1322 ◽  
Author(s):  
M Lipinski ◽  
W H Fridman ◽  
T Tursz ◽  
C Vincent ◽  
D Pious ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Surface ◽  
Target Cell ◽  
Epstein Barr Virus ◽  
Target Cells ◽  
Specific Lysis ◽  
Barr Virus ◽  
Positive Target ◽  
Epstein Barr

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.

scholarly journals Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3493-3499 ◽  
Author(s):  
BA Mehta ◽  
IG Schmidt-Wolf ◽  
IL Weissman ◽  
RS Negrin
Keyword(s):  
Cell Surface ◽  
Target Cell ◽  
Interleukin 2 ◽  
Cytoplasmic Granule ◽  
Target Cells ◽  
Killer Cells ◽  
Effector Cells ◽  
Surface Molecules ◽  
Cik Cells ◽  
Granule Release

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.

scholarly journals The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity.

1995 ◽  
Vol 181 (5) ◽  
pp. 1905-1909 ◽  
Author(s):  
H Nakajima ◽  
P Golstein ◽  
P A Henkart
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Cell Nuclei ◽  
Effector Cells ◽  
Cell Death Pathways ◽  
Mast Cell Tumors ◽  
Nuclear Damage

The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

scholarly journals CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells.

1996 ◽  
Vol 183 (6) ◽  
pp. 2533-2540 ◽  
Author(s):  
M F Krummel ◽  
J P Allison
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Apoptotic Cell ◽  
Activated T Cells ◽  
Activated State

While interactions between CD28 and members of the B7 family costimulate and enhance T cell responses, recent evidence indicates that the CD28 homologue CTLA-4 plays a downregulatory role. The mechanism by which this occurs is not clear, but it has been suggested that CTLA-4 terminates ongoing responses of activated T cells, perhaps by induction of apoptosis. Here we demonstrate that CTLA-4 engagement by antibody cross-linking or binding to B7 inhibits proliferation and accumulation of the primary T cell growth factor, IL-2, by cells stimulated with anti-CD3 and anti-CD28. This inhibition is not a result of enhanced cell death. Rather it appears to result from restriction of transition from the G1 to the S phase of the cell cycle. Our observation that upregulation of both the IL-2R alpha chain and the CD69 activation antigen are inhibited by CTLA-4 engagement supplies further evidence that CTLA-4 restricts the progression of T cells to an activated state. Together this data demonstrates that CTLA-4 can regulate T cell activation in the absence of induction of apoptotic cell death.

New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2677-2682 ◽  
Author(s):  
Inge Jedema ◽  
Nicole M. van der Werff ◽  
Renée M. Y. Barge ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Population ◽  
Target Cell ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Cell Populations ◽  
Target Cells ◽  
Specific Cell

Abstract For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

The mechanism of T cell mediated cytotoxicity V. Morphological studies by electron microscopy

1977 ◽  
Vol 198 (1132) ◽  
pp. 315-323 ◽  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Target Cell ◽  
Plasma Membranes ◽  
Ultrastructural Changes ◽  
Early Event ◽  
Target Cells ◽  
Cell Functions ◽  
Morphological Studies

The ultrastructural changes occurring when T cells specifically immune to antigens interact with P815 mastocytoma cells and EL4 lymphoma cells are described and related to changes previously observed by timelapse cinematography (Sanderson 1976 b ). In confirmation of work by others, pale T cells can clearly be in­criminated as cytotoxic cells. Dark T cells also form contacts with target cells, and tend to project lamellipodia over the surface of the target cell. The possibility is discussed that these represent a subset of non-cytotoxic, antigen-reactive T cells involved in other T cell functions. T cells form two types of contact: relatively small point contacts, and large areas where the two plasma membranes are in close apposition. No structures resembling specialized junctions or membrane fusions were observed in areas of contact between T cells and target cells. Close contact between pale T cells and target cells is more regular than contact with dark T cells. Many contacts were seen between morphologically normal target cells and pale T cells, and these were thought to occur in the phase between contact and target cell death. Some of these pale T cells in contact show projections towards the centre of the target cell which invaginate the cell membrane, but do not penetrate it. Remarkable T cell projections were also seen which had penetrated deeply through the membrane of one target cell. These projections appeared to have disrupted the mem­brane and had penetrated into apparently intact cytoplasm, suggesting that this penetration may be an early event in the lytic mechanism. The possibility that this phenomenon is the cause of cell death is discussed. Changes corresponding to the phase of zeiosis of the target cell are described. These commence with the formation of surface blebs, accompanied by a general mis-shapening of the cell outline and followed by vacuolation and loss of cytoplasmic organelles. Breakdown of the nucleus appears to be a later, post mortem event.

scholarly journals A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 399
Author(s):  
Aerin Yoon ◽  
Shinai Lee ◽  
Sua Lee ◽  
Sojung Lim ◽  
Yong-Yea Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Killing ◽  
Bispecific Antibodies ◽  
Target Cells

As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3ε. MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3ε, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

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